The Amyloid Hypothesis of Alzheimer Pathogenesis

APP

Amyloid-production

Self-assembly

clinical ADneuronal cell death

tautau

SWAN Proteins Wong & Wu

APP: Chromosomal Location

APP Genetic locus on Chr. 21q21.3

APP is a single copy gene. It exists as one copy per haploid genome.

SWAN Proteins Wong & Wu

APP is a gene that encodes a complexity of proteins

• A single locus: single copy per haploid genome• Multiple splice variant mRNAs• Multiple protein isoforms expressed

– Some isoforms are ubiquitous, others neuron specific or T cell-specific

• Variety of conserved functional domains throughout length of protein

– Isoform differences in major domains• APP interacts with multiple proteins• Mutations throughout protein associated with familial Alzheimer

Disease• Cleavage products generated by α-, β- and γ-secretase are

involved in both normal and pathogenic protein processing.

SWAN Proteins Wong & Wu

APP Gene encodes 10 Isoforms produced by Alternative Splice Variants

There are three major isoforms:

There are seven and probably more additional isoforms.

SWAN Proteins Wong & Wu

Isoform Hep Zn+/Cu+ Kunitz Hep Hep Hep β α γ Goα Casp

Isoform APP770 P05067-1 + + + + + + + + + + +Isoform APP751 P05067-8 + + + + + + + + + + +Isoform APP695 P05067-4 + + + + + + + + +Isoform L-APP677 P05067-3 + + + + + + + + + +Isoform L-APP696 P05067-5 + + + + + + + + + +Isoform L-APP733 P05067-7 + + + + + + + + + + +Isoform APP714 P05067-6 + + + + + + + + + +Isoform APP305 P05067-2 + +Isoform L-APP752 P05067-9 + + + + + + + + + + +Isoform APP639 P05067-10 + + + + + + + + + +

TMKunitzHep

Zn+/Cu+

Hep Hep Goα

Caspase

Conserved Functional Protein Domains in APP Suggest Functional Differences between Protein Isoforms

SWAN Proteins Wong & Wu

APP does more than produce Aβ…

but production of Aβ is the critical step for AD.

SWAN Proteins Wong & Wu

Intracellular Processing of APP Generates a Variety of Cleavage

Products APP is Cleaved into the following 12 chains:    1- Soluble APP-α     2- Soluble APP-β    3- C99    4- Beta-amyloid protein 42    5- Beta-amyloid protein 40    6- C83    7- P3(42): α-secretase and γ-secretase product    8- P3(40): α-secretase and γ-secretase product    9- Gamma-secretase C-terminal fragment 59        Alternative name(s):            γ-CTF(59)            Amyloid intracellular domain 59              Short name=AICD-59 or AID(59)    10- Gamma-secretase C-terminal fragment 57        Alternative name(s):            γ-CTF(57)            Amyloid intracellular domain 57              Short name=AICD-57 or AID(57)    11- Gamma-secretase C-terminal fragment 50        Alternative name(s):            γ-CTF(50)            Amyloid intracellular domain 50              Short name=AICD-50 or AID(50)    12- C31

http://journals.prous.com/journals/dnp/20061907/html/dn190411/images/fig1.gif

SWAN Proteins Wong & Wu

Intracellular APP trafficking

SWAN Proteins Wong & Wu

APP interacts with many proteins

http://string.embl.de/newstring_cgi/show_network_section.pl?taskId=TuwI5V8me0SR&use_java=no&network_flavor=evidence

APP Binds

UniProt APP Binds

UniProt

APBA1 Q02410 HSD17B10 Q99714

APBB1 O00213 MAPK8IP1

Q9UQF2

Apbb1 P46933 Mapk8ip1 Q9WVI9-1

APBB2 Q92870 MAPT P10636

Apbb3 O35827 MAPT P10636-8

APLP1 P51693 PRNP P04156

APLP2 Q06481 PSEN1 P49768

APOA1 P02647 PSEN2 P49810

CHRNA7 P36544 SHC1 P29353

Chrna7 Q05941 SHC3 Q92529

CNTN3 Q9P232 Slc5a7 Q8BGY9

CNTN4 Q8IWV2 TGFB1 P011371

FLOT1 O75955 TGFB2 P61812

HOMER2 Q9NSB8 TP53BP2 Q13625

HOMER3 Q9NSC5

APP

Amyloid- (A40 vs A42)

Amyloid Plaques

Genetic Mutations in the Gene for APP Can Cause Alzheimer’s Disease

-Secretase: Carries out the first cut that produces the N-terminus of A

Wolfe. Sci. Am., 2006

Co-crystal structure of Co-crystal structure of -secretase with an inhibitor:-secretase with an inhibitor:coordination of hydroxyl group with the catalytic aspartatescoordination of hydroxyl group with the catalytic aspartates

Hong et al. Science, 2000, 290, 150-3

BACE1 splice isoformsAlternative splicing within exons 3 and 4

BACE2Homolog of BACE1; also cleaves APP.

BACE1 substratesAPP, neuregulin, ST6Gal 1, Nav1 2, others

Processing of BACE1Signal sequence and prodomain removal, glycosylation

Intracellular localization of BACE1ER, Golgi, cell surface, endosomes

Tissue distribution of BACE1Ubiquitous but most highly in the brain

The Complexities of -Secretase (-site APP cleaving enzyme; BACE1)

-SecretasesMetalloproteases ADAM-10, -17, and -9

Where they cut within APPWithin the A sequence, thereby precluding A formation

How they are regulatedADAM-10 is constitutive, while ADAM-17 is inducible

Why they may be important therapeuticallyActivity can be stimulated by agonists for certain muscarinic receptor subtypes

The Complexities of -Secretase

Other substratesThese are major sheddases; many other membrane protein substrates

-Secretase: Performs the second cut that produces the C-terminus of A

Wolfe. Sci. Am., 2006

DD

lumen/extracellular

cytosol

Presenilin

OH

PhO

NH

Ph

NH NHO Ph

O

NH-Bt

O

O

HN N

H

HN N

HOBt

O

OH O

OF FBr

O

NTF CTF

A

AICD

APP

Wolfe et al. Nature 1999, 398, 513.Li et al. Nature 2000, 405, 689.Esler et al. Nature Cell Biology 2000, 2, 428.

p3/A

C83/C99

p6

Nicastrin,other

factors

PS1

1 2

3

Proposed Mechanism of -Secretase

Esler et al. PNAS, 2002, 99, 2720-7

2003

2001

1999

1997

SREBP Reg

S1P

S2P

lumen/extracellular

cytosol

S2P

SPP

SP

lumen/extracellular

cytosol

sig

na

l pep

tid

e

SPP

S4

S2,

DD

lumen/extracellular

cytosol

Aph-1 NCT

CTF

NTF

Pen-2

Presenilin

S3,

Spitz

RhomboidS H

lumen/extracellular

cytosol

Rhomboid

Wolfe and Kopan. Science 2004

DD

HE

XX

H

LD

G

Aph-1

NCT

NTF

Pen-2

-Secretase: A Complex and Unusual Protease

Takasugi et al. Nature 2003Kimberly et al. PNAS 2003Edbauer et al. Nat Cell Biol 20031:1:1:1 Stoichiometry: Sato et al. JBC 2007

CTF

The size of the -secretase complex is ~230 kDa by scanning transmission electron microscopy

Osenkowski et al. JMB 2009.

CryoEM Structure of -Secretase

Osenkowski et al. JMB 2009.

PS mutationsOver 100 FAD-associated mutations

PS1 vs PS2PS1 is the major player, both in AD and biology

Aph1 isoformsAph1- and Aph1-ph1- and Aph1-2

Nicastrin glycosylation16 glycosylation sites that occur in transit from the ER

Other processing events at PSCaspase cleavage gives short CTF

Intracellular localizationER, Golgi, cell surface, endosomes, mitochondria

The Complexities of -Secretase

S4

S2

DD

lumen/extracellular

cytosol

Presenilin

S3

NTF CTF

Np3

NICD

Notch

DeStrooper et al. Nature 1999, 398, 518.

AP

P

AICD

A48/49

A45/46 A42/43

’

A38/40

Ihara Model of Processive Proteolysis of APP

When Tau Goes Bad

Wolfe. Sci. Am., 2006

SWAN Proteins Wong

Tau: Chromosomal LocationMAPT Genetic locus on Chr. 17q21.1

WT: MAPT is a single copy gene. It exists as one copy per haploid genome. Disease: MAPT gene mutations have been associated with several neurodegenerative disorders such as Alzheimer's disease, Pick's disease, frontotemporal dementia, cortico-basal degeneration and progressive supranuclear palsy.

Source: MAPT Mapviewer (NCBI)

SWAN Proteins Wong

Tau is a gene that encodes a complexity of proteins

• A single locus: single copy per haploid genome

• Multiple splice variant mRNAs

• Mulitiple protein isoforms expressed– tau is not only present in neurons but is also clearly present in

oligodendroglia and astrocytes

• Variety of conserved functional domains throughout length of protein

– Isoform differences in major domains

• Tau interacts with multiple proteins

• ~40 FTDP-associated mutations

Alternative Splicing of Tau in FTDP-17

Wolfe. JBC 2009

Stem Loop Structure and Exon 10 Splicing

Wolfe. JBC 2009

SWAN Proteins Wong

Tau protein is highly phosphorylated: Phosphorylation state appears to be critical to disease

Acknowledgments

Funding

National Institutes of HealthAlzheimer’s Association

ISOA/ADDF

Wolfe Lab

ProteasesWilliam EslerTaylor KimberlyChittaranjan DasFrédéric BihelAnna KornilovaPatrick FraeringPam OsenkowskiToru SatoSarav NarayananOmar Quintero-MonzonMorgan MartinMarty Fernandez

RNAChristine DonahueYang LiuEleanor PeaceyKaren MowrerJean-François Fisette

Collaborators

ProteasesDennis SelkoeRafi Kopan

Bart De StrooperTodd Golde

Huilin Li

RNAKen KosikJane Wu

Eriks RosnersMarcie GlicksmanGabriele Varani

LEAD

Corinne Augelli-SzafranHan-Xu Wei

Dai LuJing ZhangYongli Gu